WO2012086474A1

WO2012086474A1 - Therapeutic agent for central nervous system diseases

Info

Publication number: WO2012086474A1
Application number: PCT/JP2011/078846
Authority: WO
Inventors: 健治門松; 史郎今釜; 亮吏田内
Original assignee: 国立大学法人名古屋大学
Priority date: 2010-12-21
Filing date: 2011-12-14
Publication date: 2012-06-28
Also published as: JPWO2012086474A1

Abstract

The purpose of the present invention is to provide a novel therapeutic means for central nervous system diseases. Provided is a therapeutic agent for central nervous system diseases, said therapeutic agent comprising, as the active ingredient, ADAMTS-4 protein or an expression vector carrying ADAMTS-4 gene.

Description

CNS therapeutic agent

The present invention relates to a therapeutic agent for central nervous disease and its use. This application claims priority based on Japanese Patent Application No. 2010-283910 filed on Dec. 21, 2010, the entire contents of which are incorporated by reference.

Conventionally, it has been said that the central nerve once damaged is not regenerated. In fact, once the central nerves, such as the brain and spinal cord, are damaged by trauma or hemorrhage, and neuropathy appears, it is not usually cured. The reason for this is thought to be a factor that suppresses it compared to a factor that promotes regeneration. Among them, the myelin-related proteins Nogo, MAG, OMgp and chondroitin sulfate proteoglycan (hereinafter “CSPG”) are well known as nerve regeneration inhibitors. In 2002, Bradburry et al. Reported that hindlimb motor function was restored by administering chondroitinase ABC (hereinafter referred to as “C-ABC”), which degrades CS chain of CSPG, to the injured spinal cord of rats (Non-patent Document 1). ). Similarly, there is a keratan sulfate proteoglycan (hereinafter referred to as “KSPG”) that accumulates around the injury after spinal cord injury and suppresses nerve regeneration. Our group reported that when spinal cord injury occurred in a KSPG knocked out mouse, the hindlimb motor function after the injury was restored compared to the wild type mouse (Non-patent Document 2).

An object of the present invention is to provide a novel therapeutic means for central nervous system diseases.

Under the above problems, the present inventors proceeded with research. First, as with C-ABC, Keratanase II (K-II), an enzyme that degrades keratan sulfate after spinal cord injury, was administered into the subarachnoid space of a spinal cord injury model (rat). A similar recovery was obtained. However, co-administration of C-ABC and K-II was performed, and the effect at that time was equivalent to that of C-ABC alone and K-II alone. The reason why the additional effect was not obtained was suggested that the structural change of proteoglycan due to the CS chain of CSPG and the KS chain of KSPG being broken affects the elongation effect of nerve axon. Therefore, we focused on ADAMTS-4 (A disintegrin and metalloprotease with thrombospondin motif 4), which degrades the core protein, as an enzyme that causes similar structural changes in proteoglycans. In other words, the experiment was carried out on the assumption that the structural change of proteoglycan caused by the degradation of core protein by ADAMTS-4 has the effect of elongating nerve axons after spinal cord injury. As a result, ADAMTS-4 showed a high nerve axon extension effect and was found to be extremely effective in improving function after spinal cord injury. In addition, after spinal cord injury, although the expression level of ADMATS4 does not change at the mRNA level, an increase is seen at the protein level, and ADAMTS-4 is highly expressed at astrocytes and microglia rather than neurons, Furthermore, important and useful findings were obtained regarding the mechanism of action of ADAMTS-4 in the regeneration of the central nervous system, such as the degradation of proteoglycans in the brain, such as blebican, neurocan and phosphacan, which occurs specifically in ADMATS4.

As described above, as a result of intensive studies by the present inventors, the knowledge that ADAMTS-4 is extremely effective for the regeneration and healing of the central nervous system has been brought about. The present invention described below is mainly based on the findings.
[1] A therapeutic agent for central nervous disease comprising the following (1) or (2) as an active ingredient:
(1) ADAMTS-4 protein;
(2) An expression vector carrying the ADAMTS-4 gene.
[2] The therapeutic agent for central nervous disease according to [1], wherein the ADAMTS-4 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to the amino acid sequence.
[3] The therapeutic agent for central nervous disease according to [1], wherein the ADAMTS-4 gene comprises the base sequence shown in SEQ ID NO: 2 or a base sequence equivalent to the base sequence.
[4] The central nervous system disease is selected from the group consisting of spinal cord injury, myelitis, brain injury, neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, and retinal disease with neuronal damage The therapeutic agent for central nervous disease according to any one of [1] to [3], which is a disease or a disease state.
[5] The therapeutic agent for central nervous disease according to any one of [1] to [3], wherein the central nervous disease is spinal cord injury.
[6] A method for treating central nervous disease, comprising the step of administering a therapeutic agent for central nervous disease comprising the following (1) or (2) as an active ingredient to a patient with central nervous disease:
(1) ADAMTS-4 protein;
(2) An expression vector carrying the ADAMTS-4 gene.
[7] The central nervous disease is selected from the group consisting of spinal cord injury, myelitis, brain injury, neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia and intracerebral hemorrhage, and retinal disease accompanied by neuronal damage The treatment according to [6], which is a disease.
[8] The treatment method according to [6], wherein the central nervous disease is spinal cord injury.

Results of RT-PCR using mRNA extracted from damaged spinal cord tissue. The expression level of ADAMTS-4 mRNA after 3 days, 1 week and 2 weeks after spinal cord injury surgery was examined. Results of real-time RT-PCR using mRNA extracted from damaged spinal cord tissue. The expression level of ADAMTS-4 mRNA was quantified 3 days after surgery for spinal cord injury, 1 week later, and 2 weeks later. NS: No significant difference. Results of Western blot using a homogenate of damaged spinal cord tissue. One week after the operation, the ADAMTS-4 protein expression level was compared between the spinal cord injury surgery group and the sham group (10th thoracic laminectomy only). Fluorescence assay results. ADAMTS-4 activity in the injured spinal cord was measured with a fluorescence assay (n = 9). *: <0.001 Results of RT-PCR using mRNA extracted from nervous system cells. The ADAMTS-4 expression levels of neurons, astrocytes and microglia were compared. Results of degradation assay using proteoglycan (PG) collected from rat brain. The degradation activity of blebican by ADAMTS-4 and ADAMTS-13 was comparatively evaluated by Western blot. From left to right, first lane: control (PG only), second lane: ADAMTS-4 (1 μM) added, third lane: ADAMTS-4 boiled at 95 ° C, fourth lane: ADAMTS-13 ( 1 μM), 5th lane: ADAMTS-13 boiled at 95 ° C. is added. Results of degradation assay using proteoglycan (PG) collected from rat brain. Neurocan degradation activity by ADAMTS-4 and ADAMTS-13 was evaluated by Western blot. From left to right, first lane: control (PG only), second lane: ADAMTS-4 (1 μM) added, third lane: ADAMTS-4 boiled at 95 ° C, fourth lane: ADAMTS-13 ( 1 μM), 5th lane: ADAMTS-13 boiled at 95 ° C. is added. Results of degradation assay using proteoglycan (PG) collected from rat brain. The degradation activity of phosphacan by ADAMTS-4 and ADAMTS-13 was compared and evaluated by Western blot. From left to right, first lane: control (PG only), second lane: ADAMTS-4 (1 μM) added, third lane: ADAMTS-4 boiled at 95 ° C, fourth lane: ADAMTS-13 ( 1 μM), 5th lane: ADAMTS-13 boiled at 95 ° C. is added. Results of axon elongation assay. The axon extension effect of ADAMTS-4 was investigated in the presence and absence of PG. PLL: Poly-L-lysine-coated slide used (no enzyme added), ADAMTS-4 + PLL: Poly-L-lysine-coated slide and ADAMTS-4 added, ADAMTS-13 + PLL: Poly-L-lysine-coated slide used ADAMTS-13 added, PG: using proteoglycan coated slide (no enzyme added), ADAMTS-4 + PG: using proteoglycan coated slide and ADAMTS-4 added, ADAMTS-13 + PG: using proteoglycan coated slide and ADAMTS-13 added, C- ABC + PG: Use proteoglycan-coated slides and add C-ABC. NS: No significant difference. *: <0.01. Results of axon elongation assay. The number of adherent cells was compared under each condition. PG: Proteoglycan coated slide use (without enzyme addition), ADAMTS-13: Proteoglycan coated slide use and ADAMTS-13 addition, ADAMTS-4: Proteoglycan coated slide use and ADAMTS-4 addition, C-ABC: Proteoglycan coated Use slide and add C-ABC. *: <0.001. Results of hindlimb motor function evaluation using the BBB scale. Rats subjected to spinal cord injury surgery were administered ADAMTS-4, c-ABC, or a drug solution containing only vehicle (Vehicle) into the subarachnoid space, and the hindlimb motor function was compared and evaluated over time (n = 6). *: <0.05. Results of immunohistochemical staining of 5-HT fibers. Upper left, lower left and upper right are stained images. The lower right is the measurement result of 5HT positive area.

1. Therapeutic Agent for Central Nervous Disease The first aspect of the present invention relates to a therapeutic agent for central nervous disease (hereinafter also referred to as “the therapeutic agent of the present invention” for convenience of explanation). Although the nervous system is roughly classified into a central nervous system and a peripheral nervous system, the present invention provides a therapeutic agent applied to diseases of the central nervous system. As used herein, “therapeutic agent for central nervous system disease” refers to a drug used for the treatment of central nervous system diseases.

The therapeutic agent of the present invention contains (1) ADAMTS-4 protein or (2) an expression vector carrying the ADAMTS-4 gene as an active ingredient. In addition, although the therapeutic agent of this invention contains either (1) and (2) normally, it does not prevent containing these both.

(1) ADAMTS-4 protein ADAMTS-4 (A disintegrin and metalloprotease with thrombospondin motif 4) is a molecule belonging to the ADAMTS family and is also called aggrecanase. The amino acid sequence of ADAMTS-4 protein (GenPept (NCBI), ACCESSION: NP_005090, DEFINITION: A disintegrin and metalloproteinase with thrombospondin motifs 4 preproprotein [Homo sapiens]) registered in the public database is shown in SEQ ID NO: 1 Show. A part of ADAMTS-4 protein (partial protein) may be used as long as the intended effect of the present invention, that is, effectiveness against central nervous system diseases is maintained. As an example of such a partial protein, its amino acid sequence is shown in SEQ ID NO: 3 (sequence corresponding to amino acid residues 52 to 685). In the present specification, “ADAMTS-4 protein” is used as a term including such partial proteins.

A polypeptide containing an amino acid sequence equivalent to the amino acid of SEQ ID NO: 1 or the amino acid of SEQ ID NO: 3 can also be used as the ADAMTS-4 protein. The “equivalent amino acid sequence” here is partly different from the reference amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 3), but the difference is the function of the protein (effective action against central nervous disease). ) Refers to an amino acid sequence that does not substantially affect Therefore, substantial identity is recognized between the reference amino acid sequence (SEQ ID NO: 1 or SEQ ID NO: 3) and the equivalent amino acid sequence. In order to determine the presence or absence of substantial identity, for example, using the experimental system (evaluation by an animal model) described in the examples below, between the two amino acid sequences in terms of the action / effect on the central nervous system disease It is sufficient to confirm that there is no substantial difference between the two. ADAMTS-4 is highly conserved and ADAMTS-4 has been identified in various animal species.

“Different in part of amino acid sequence” typically means deletion or substitution of 1 to several amino acids (upper limit is 3, 5, 7, 10) constituting the amino acid sequence. Alternatively, it means that a mutation (change) has occurred in the amino acid sequence due to addition, insertion, or a combination of 1 to several amino acids (the upper limit is, for example, 3, 5, 7, 10). Differences in amino acid sequences here are allowed as long as there is no significant decrease in the above functions. As long as this condition is satisfied, the positions where the amino acid sequences are different are not particularly limited, and differences may occur at a plurality of positions. The term “plurality” as used herein refers to, for example, a number corresponding to less than about 30% of all amino acids, preferably a number corresponding to less than about 20%, and more preferably a number corresponding to less than about 10%. The number is preferably less than about 5%, and most preferably less than about 1%. That is, the equivalent amino acid sequence is the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence of SEQ ID NO: 3), for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, and still more preferably about 95%. As described above, the sequence identity of about 99% or more is most preferable.

It is preferable that the difference between the reference amino acid sequence (SEQ ID NO: 1, SEQ ID NO: 3) and the equivalent amino acid sequence is caused by a conservative amino acid substituent. As used herein, “conservative amino acid substitution” refers to substitution of a certain amino acid residue with an amino acid residue having a side chain having the same properties. Depending on the side chain of the amino acid residue, a basic side chain (eg lysine, arginine, histidine), an acidic side chain (eg aspartic acid, glutamic acid), an uncharged polar side chain (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine), aromatic side chains (eg tyrosine, phenylalanine, Like tryptophan and histidine). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

Incidentally, the sequence identity (%) of two amino acid sequences or two nucleic acid sequences (hereinafter, “two sequences” is used as a term including them) can be determined, for example, by the following procedure. First, two sequences are aligned for optimal comparison (eg, a gap may be introduced into the first sequence to optimize alignment with the second sequence). When a molecule (amino acid residue or nucleotide) at a specific position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is the same. Sequence identity is a function of the number of identical positions common to the two sequences (ie, sequence identity (%) = number of identical positions / total number of positions × 100), preferably for alignment optimization Take into account the number and size of gaps required.

比較 Comparison of two sequences and determination of identity can be achieved using a mathematical algorithm. Specific examples of mathematical algorithms that can be used for sequence comparison are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 226 87: 2264-68; Karlin and Altschul (1993) Proc. Natl. There is a modified algorithm in Acad. Sci. USA 90: 5873-77, but it is not limited to this. Such an algorithm is incorporated in the NBLAST program and XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule in the present invention, for example, a BLAST nucleotide search may be performed with the score = 100 and wordlength = 12 in the NBLAST program. In order to obtain an amino acid sequence equivalent to the reference amino acid sequence, for example, a BLAST polypeptide search may be performed using the XBLAST program with score = 50 and wordlength = 3. In order to obtain a gap alignment for comparison, Gapped BLAST described in Altschul et al. (1997) Amino Acids Research 25 (17): 3389-3402 can be used. When utilizing BLAST and Gapped BLAST, the default parameters of the corresponding programs (eg, XBLAST and NBLAST) can be used. For details, refer to the NCBI web page. Examples of other mathematical algorithms that can be used for sequence comparison include those described in Myers and Miller (1988) Comput Appl Biosci. 4: 11-17. Such an algorithm is incorporated in the ALIGN program available on, for example, the GENESTREAM network server (IGH （Montpellier, France) or the ISREC server. When using the ALIGN program for comparison of amino acid sequences, for example, a PAM120 residue mass table can be used, with a gap length penalty = 12 and a gap penalty = 4.

The identity of two amino acid sequences, using the Gloss program in the GCG software package, using a Blossom 62 matrix or PAM250 matrix, gap weight = 12, 10, 8, 6, or 4, gap length weight = 2, 3 Or 4 can be determined. Also, the identity of two nucleic acid sequences can be determined using the GAP program of the GCG software package, with gap weight = 50 and gap length weight = 3.

ADAMTS-4 protein is easily prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. can do. For example, it can be prepared by transforming a suitable host cell (for example, E. coli, yeast) with DNA encoding ADAMTS-4 protein and recovering the protein expressed in the transformant. The recovered protein is appropriately purified according to the purpose. Thus, various modifications are possible if ADAMTS-4 protein is obtained as a recombinant protein. For example, if a DNA encoding ADAMTS-4 protein and other appropriate DNA are inserted into the same vector and a recombinant protein is produced using the vector, a recombinant protein in which any peptide or protein is linked ADAMTS-4 protein consisting of can be obtained. In addition, modification may be performed so that addition of sugar chain and / or lipid, or processing of N-terminal or C-terminal may occur. By the modification as described above, extraction of recombinant protein, simplification of purification, addition of biological function, and the like are possible. The ADAMTS-4 protein is also commercially available (for example, recombinant human ADAMTS-4 (having the amino acid sequence of SEQ ID NO: 3) provided by R & D® Systems, Inc., catalog number 4307-AD) and is readily available. It is.

In view of qualitative uniformity and purity, ADAMTS-4 protein is preferably prepared by genetic engineering techniques. However, the method for preparing ADAMTS-4 protein is not limited to genetic engineering. For example, ADAMTS-4 protein can be prepared from natural materials by standard techniques (crushing, extraction, purification, etc.).

(2) Expression Vector Holding ADAMTS-4 Gene In one embodiment of the present invention, an expression vector holding ADAMTS-4 gene (hereinafter referred to as “ADAMTS-4 gene”) is used as an active ingredient. “Expression vector” refers to a vector capable of introducing a nucleic acid inserted therein into a target cell (host cell) and allowing expression in the cell. In the expression vector according to the present invention, the ADAMTS-4 gene is retained so that it can be expressed. The type of the vector is not particularly limited as long as the ADAMTS-4 gene can be introduced into the target cell and expressed in the target cell. The “vector” herein includes viral vectors and non-viral vectors. The gene transfer method using a virus vector skillfully utilizes the phenomenon that a virus infects cells, and high gene transfer efficiency can be obtained. Adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, herpes virus vectors, Sendai virus vectors and the like have been developed as virus vectors.

Non-viral vectors include liposomes, positively charged liposomes (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84: 7413-7417, 1987), HVJ (Hemagglutinating virus of Japan) -Liposome (Dzau, VJ, Mann, M., Morishita, R. et al., Proc. Natl. Acad. Sci., 93: 11421-11425, 1996, Kaneda, Y. , R., Molecular Med. Today, 5: 298-303, 1999). The vector in the present invention may be constructed as such a non-viral vector. Further, a YAC vector, a BAC vector or the like may be used.

In adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, a foreign gene incorporated into the vector is incorporated into the host chromosome, and stable and long-term expression can be expected. Retroviral vectors are not suitable for gene transfer into non-dividing cells because cell division is required for integration of the viral genome into the host chromosome. On the other hand, lentivirus vectors and adeno-associated virus vectors cause integration of foreign genes into the host chromosome after infection even in non-dividing cells. Therefore, these vectors are effective for stably and long-term expressing foreign genes in non-dividing cells.

Each virus vector can be prepared according to a previously reported method or using a commercially available dedicated kit. For example, an adenovirus vector can be prepared by the COS-TPC method or full-length DNA introduction method. The COS-TPC method is a homologous recombination that occurs in 293 cells by co-transfecting a recombinant cosmid incorporating the target cDNA or expression cassette and a parent virus DNA-terminal protein complex (DNA-TPC) into 293 cells. (Mayake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA introduction method is a method for producing a recombinant adenovirus by subjecting a recombinant cosmid inserted with a target gene to restriction digestion and then transfecting 293 cells (Miho Terashima, Koki Kondo). Hiromi Kanegae, Izumi Saito (2003) Experimental Medicine 21 (7) 931.). The COS-TPC method can be performed using Adenovirus® Expression® Vector® Kit® (Dual® Version) (Takara Bio Inc.) and Adenovirus® genome® DNA-TPC (Takara Bio Inc.). In addition, the full-length DNA introduction method can be performed using Adenovirus® Expression® Vector® Kit® (Dual® Version) (Takara Bio Inc.).

On the other hand, retroviral vectors can be prepared by the following procedure. First, the virus genome (gag, pol, env gene) other than the packaging signal sequence between the LTRs (Long Terminal Repeat) existing at both ends of the virus genome is removed, and the target gene is inserted therein. The viral DNA thus constructed is introduced into a packaging cell that constitutively expresses the gag, pol, and env genes. Thereby, only the vector RNA having the packaging signal sequence is incorporated into the viral particle, and a retroviral vector is produced.

Developed by improving or specificizing fiber protein by modifying adeno vectors (specific infection vectors) and gutted vectors (helper-dependent vectors) that can be expected to improve the expression efficiency of target genes. Has been. The expression vector of the present invention may be constructed as such a viral vector.

The ADAMTS-4 gene inserted into the vector (hereinafter referred to as “ADAMTS-4 gene”) is, for example, SEQ ID NO: 2 (GenBank (NCBI), ACCESSION: NM_005099, DEFINITION: Homo sapiens ADAM metallopeptidase with thrombospondin type 1 motif, 4 (ADAMTS -4), mRNA.) Or SEQ ID NO: 4 (sequence encoding amino acid residues 53 to 685 of the amino acid sequence of SEQ ID NO: 1). However, DNA having a base sequence equivalent to the base sequence (hereinafter referred to as “equivalent DNA”) can also be used as the ADAMTS-4 gene. The “equivalent base sequence” here is partially different from the standard base sequence, but the function of the protein encoded by this difference (effective action on central nervous disease) has a substantial effect. The base sequence that has not been received. A specific example of equivalent DNA is DNA that hybridizes under stringent conditions to a base sequence complementary to a reference base sequence (for example, the base sequence of SEQ ID NO: 2 or SEQ ID NO: 4). The “stringent conditions” here are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Such stringent conditions are known to those skilled in the art, such as Molecular Cloning (Third Edition, Cold Spring Harbor Laboratory Press, New York) and Current protocols in molecular biology (edited by Frederick M. Ausubel et al., 1987) Can be set with reference to. As stringent conditions, for example, hybridization solution (50% formamide, 10 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 5 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml denaturation Conditions of incubation at about 42 ° C to about 50 ° C using salmon sperm DNA, 50 mM phosphate buffer (pH 7.5), followed by washing at about 65 ° C to about 70 ° C using 0.1 x SSC, 0.1% SDS Can be mentioned. Further preferable stringent conditions include, for example, 50% formamide, 5 × SSC (0.15M NaCl, 15 mM sodium citrate, pH 7.0), 1 × Denhardt solution, 1% SDS, 10% dextran sulfate, 10 μg / ml as a hybridization solution. Of denatured salmon sperm DNA, 50 mM phosphate buffer (pH 7.5)).

As another specific example of equivalent DNA, consisting of a base sequence including substitution, deletion, insertion, addition, or inversion of one or more bases with respect to a reference base sequence (SEQ ID NO: 2, SEQ ID NO: 4), A DNA encoding a protein effective against non-ischemic myocardial injury can be mentioned. Base substitution or deletion may occur at a plurality of sites. The term “plurality” as used herein refers to, for example, 2 to 40 bases, preferably 2 to 20 bases, more preferably 2 to 10 bases, although it varies depending on the position and type of amino acid residues in the three-dimensional structure of the protein encoded by the DNA. It is. Such equivalent DNAs include, for example, restriction enzyme treatment, treatment with exonuclease and DNA ligase, position-directed mutagenesis (MolecularMCloning, Third Edition, Chapter 13, Cold Spring Harbor Laboratory Press, New York) Includes substitutions, deletions, insertions, additions, and / or inversions of bases using mutation introduction methods (Molecular Cloning, ingThird Edition, Chapterhap13, Cold Spring Harbor Laboratory Press, New York) Thus, it can obtain by modifying DNA which has a standard base sequence. The equivalent DNA can also be obtained by other methods such as ultraviolet irradiation.

As yet another example of equivalent DNA, there can be mentioned DNA in which a difference in base as described above is recognized due to a polymorphism represented by SNP (single nucleotide polymorphism).

The ADAMTS-4 gene can be prepared by using standard genetic engineering techniques, molecular biological techniques, biochemical techniques, etc. with reference to the sequence information disclosed in this specification or the attached sequence listing. it can. For example, the ADAMTS-4 gene can be isolated (and amplified) from a human cDNA library by appropriately using an oligonucleotide probe / primer that can specifically hybridize to the ADAMTS-4 gene. As the oligonucleotide probe primer, for example, a DNA complementary to the base sequence shown in SEQ ID NO: 2 or the base sequence shown in SEQ ID NO: 4 or a continuous part thereof is used. Oligonucleotide probes and primers can be easily synthesized using a commercially available automated DNA synthesizer. For the method of preparing a library used for preparing the ADAMTS-4 gene, for example, Molecular® Cloning, • Third • Edition, • Cold®Spring®Harbor®Laboratory®Press, and “New York” are helpful.

An equivalent DNA can be prepared by using a cDNA library derived from a non-human mammalian cell (for example, monkey, mouse, rat, pig, bovine) instead of the human cDNA library.

Preparation of the therapeutic agent of the present invention can be performed according to a conventional method. In the case of formulating, other pharmaceutically acceptable ingredients (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological Saline solution and the like). As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the soothing agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. As the stabilizer, propylene glycol, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As preservatives, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used. Antibiotics, pH adjusters, growth factors (eg, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)), and the like may be included.

When an expression vector carrying the ADAMTS-4 gene is used as an active ingredient, it may be formulated in combination with a pharmaceutically acceptable medium. “Pharmaceutically acceptable medium” refers to a substance that provides advantages or benefits with respect to administration or storage of an expression vector without substantially affecting the efficacy of the expression vector (ie, efficacy against central nervous system diseases). . Examples of the “pharmaceutically acceptable medium” include deionized water, ultrapure water, physiological saline, phosphate buffered saline (PBS), and 5% dextrose aqueous solution.

When the expression vector carrying the ADAMTS-4 gene is in the form of a viral vector, it is preferable to use a biocompatible polyol (for example, poloxamer 407) in combination. The use of polyols can increase the transduction rate of viral vectors by 10 to 100 times (March et al., Human Generap Therapy 6: 41-53,） 1995). Therefore, if a polyol is used in combination, the dose of the viral vector can be kept low. In addition, you may decide to use a polyol as one component of the therapeutic agent of this invention, or to prepare a polyol (or composition containing it) separately from the therapeutic agent of this invention. In the latter case, when the therapeutic agent of the present invention is administered, polyol (or a composition containing it) is also administered.

The dosage form for formulation is not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, and suppositories.

The therapeutic agent of the present invention contains an active ingredient in an amount necessary for obtaining an expected therapeutic effect (or preventive effect) (that is, a therapeutically effective amount). The amount of the active ingredient in the therapeutic agent of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, within the range of about 0.1 wt% to about 99 wt% so as to achieve a desired dose.

As long as it is delivered to the target tissue, the administration route of the therapeutic agent of the present invention is not particularly limited. For example, it is applied by topical administration. Examples of topical administration include injection or application to the target tissue. When applied to spinal cord injury, for example, it may be injected into the subarachnoid space. The therapeutic agent of the present invention may be administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. These administration routes are not mutually exclusive, and two or more arbitrarily selected can be used in combination.

The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quails, etc.). In a preferred embodiment, the therapeutic agent of the present invention is applied to humans. Preferably, the composition of the present invention is administered to a subject in the acute phase or the subacute phase so as to maximize the effect of the therapeutic agent of the present invention.

The dose of the therapeutic agent of the present invention is set so as to obtain the expected therapeutic effect. In setting a therapeutically effective dose, the patient's symptoms, age, sex, weight, etc. are generally considered. A person skilled in the art can set an appropriate dose in consideration of these matters. For example, when ADAMTS-4 protein is used as an active ingredient, for example, for an adult (body weight of about 60 kg), the dose is adjusted so that the amount of active ingredient per day is about 20 μg to about 20 mg, preferably about 2 mg to about 10 mg. Can be set. As the administration schedule, for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the patient's symptoms and the duration of effect of the active ingredient can be taken into consideration.

The therapeutic agent of the present invention is used for the treatment of central nervous disease. Without being bound by theory, according to the therapeutic agent of the present invention, the active ingredient causes a structural change of proteoglycan that inhibits regeneration of the damaged central nerve, and exhibits a therapeutic effect. If the treatment based on such a mechanism is an effective disease or pathological condition, it can be a target disease of the present invention regardless of its type or cause. Preferably, central nervous diseases caused by trauma or bleeding, specifically spinal cord injury, myelitis, brain injury, neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, and neuronal damage The therapeutic agent of the present invention is applied to retinal diseases and the like involving

Spinal cord injury refers to a condition in which the spinal cord is damaged by an external impact or an internal factor such as a spinal cord tumor or hernia. Depending on the degree of injury, it is divided into a complete type (a state in which the spinal cord is completely cut halfway) and an incomplete type (a state in which the spinal cord is damaged or compressed, but the function of the spinal cord is partially maintained). Current medical technology cannot completely recover spinal cord injury, and the establishment of a new treatment is eagerly desired. Spinal cord injury is one of the diseases for which regenerative medicine is expected to be applied, and the use of bone marrow, neural stem cells, embryonic stem cells, induced pluripotent stem cells and the like has been studied. However, due to various problems, a definitive treatment technique has not been realized. The therapeutic agent of the present invention provides a therapeutic method that can be expected to have a high therapeutic effect in such a situation, and its significance and value are extremely high.

Suppose that the structural change of proteoglycan caused by the degradation of core protein by ADAMTS-4 brings about the effect of elongating nerve axons after spinal cord injury, the following experiment was conducted.

1. Method (1) Surgery for spinal cord injury Female SD (Sprague-Dawley) rats (200-230 g) were used. Ketamine (100 mg / kg) and xylazine (10 mg / kg) were administered intraperitoneally to rats and anesthetized. A tenth thoracic laminectomy was performed to expose the dura mater. Spinal cord injury was created with an intensity of 200 kdyn using IH impactor (Infinite Horizon impactor; Precision Systems & Instrumentation, Lexington, KY). The eleventh thoracic vertebral arch was partially excised, and a silicon tube connected to a pump (Alzet pump) into which a drug solution had been previously injected was placed in the subarachnoid space under a microscope. Manual urination was performed twice a day. Food was placed at the bottom of the cage and a drinking bottle with antibiotics was installed. Management was performed according to the guidelines of the Nagoya University School of Medicine.

(2) Measurement of ADAMTS-4 mRNA in the injured spinal cord (RT-PCR and real-time PCR)
Using the RNeasy Lipid Tissue Mini Kit (QIAGEN), mRNA was extracted from the damaged spinal cord tissue according to the protocol. CDNA was prepared from mRNA using SuperScriptIII First-Strand Synthesis SuperMix (Invitrogen). The following primers were used for PCR.
<Primer for ADAMTS-4>
5'-ctacaaccaccgaaccgac-3 '(SEQ ID NO: 5)
5'-tgccagccaccagaactt-3 '(SEQ ID NO: 6)
<Rat-specific GAPDH primer 5'-tatgactctacccacggcaag-3 '(SEQ ID NO: 7)
5'-tgcattgctgacaatcttgag-3 (SEQ ID NO: 8)

Real-time PCR was performed to quantify ADAMTS-4 mRNA during spinal cord injury. Real-time PCR was performed using a Light Cycler 480 Real-Time System (Roche Diagnostics Corporation) and a Light Cycler Fast Start DNA Master SYBR Green I (Roche Diagnostics Corporation). The following primers were used in real-time PCR.
<Rat-specific ADAMTS-4 primer>
5'-cccggaatggtggaaagtatt-3 '(SEQ ID NO: 9)
5'-tcttcacggaaggtcaatgct-3 '(SEQ ID NO: 10)
<Rat specific GAPDH primer>
5'-tgattctacccacggcaagt-3 '(SEQ ID NO: 11)
5'-agcatcaccccatttgatgt-3 '(SEQ ID NO: 12)

(3) Measurement of ADAMTS-4 protein expression and activity in the injured spinal cord After spinal cord tissue homogenization at 1 week after spinal cord injury, centrifugation was performed at 10,000 g for 15 minutes. The supernatant was collected, protein quantification was performed, and Western blotting was performed with a protein amount of 20 μg. The primary antibody is anti-ADAMTS-4 (1000-fold diluted; Santa Cruz), anti-β-actin antibody (100,000-fold diluted; Sigma), the secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG antibody (5000-fold diluted) and Anti-rabbit IgG antibody (5000-fold dilution; Invitrogen) was used. ECL plus Western blotting detection kit (GE Healthcare, Buckinghamshire, UK) was used for color development. ADAMTS-4 activity in the injured spinal cord was measured by a fluorescence assay using SensoLyte (registered trademark) 520 Aggrecanase-1 assay kit (AnaSpec, San Jose, USA).

(4) Cell culture
(a) Neurons After removing the buffy coat from the cerebellum of p8 rat, it was cultured at 37 ° C. for 15 minutes. Thereafter, 10% FBS, 1M MgCl ₂ and DAase I were administered to the solution, and the cells were cultured again at 37 ° C. for 10 minutes. This was washed with PBS, placed in a container containing 60% and 35% percoll, and centrifuged. This separates neurons and glia. The sorted neurons were cultured on a petri dish.
(b) Astrocytes After removing the buffy coat from the cerebrum of p3 rat, it was cultured in a flask with DMEM containing 10% FBS under conditions of 37 ° C. and 5% CO ₂ . The culture solution was changed every 3-5 days and cultured until confluent. It was confirmed that 95% were GFAP positive cells. When the mRNA was finally collected, the microglia was removed by shaking.
(c) Microglia In the same way as astrocytes, the cell population (glia cells) is cultured for 21 days, shaken at 200 rpm for 30 minutes with a shaker, and the cells (microglia) are peeled off and collected. Cultured and used. It was confirmed that 95% or more of the cells were Iba-1 positive cells.

(5) Expression of ADAMTS-4 mRNA in neurons / astrocytes / microglia mRNA was extracted from cells using the RNeasy Mini Kit (QIAGEN) according to the attached protocol. The expression of ADAMTS-4 mRNA was confirmed by a method similar to the method (2) described above.

(6) Confirmation of proteoglycan degradation action of ADAMTS-4 by degradation assay Collecting proteoglycan (PG) of 100kDa or more from rat brain by the method of Matsui et al. (2006, Adv Pharmacol 2006; 53: 3-20) did. Add purified enzyme (ADAMTS-4 (1 μM), ADAMTS-13 (1 μM), ADAMTS-4 boiled at 95 ° C., or ADAMTS-13 boiled at 95 ° C.) to purified PG (3 μg). Incubated for hours. Then, C-ABC 10U / ml was administered, and a solution incubated at 37 ° C. for 1 hour was used as a sample, using an anti-Brevican antibody, an anti-Neurocan antibody, and an anti-Phosphacan antibody. Western blotting was performed to examine the degradation activity of each enzyme.

(7) Axon extension assay by ADAMTS-4 Neurons were collected by the method described above. 20 μg / ml poly-L-lysine (PLL; Sigma) or 300 ng / ml proteoglycan (Millipore Bioscience Research Reagents) was added dropwise to a 4-well camber slide at 4 ° C. overnight. Neurons were seeded on the slides thus prepared at a cell density of 2.0 × 10 ⁵ / well. Thereafter, 10 nM / ml ADAMTS-4, 10 U / ml C-ABC or 10 nM ADAMTS-13 was administered, and fixed with 4% paraformaldehyde 24 hours later, and nerve axon extension using anti-β-tubulin antibody The number of cell adhesion was measured.

(8) Evaluation of hindlimb motor function Rats (ADAMTS-4 (5.3 μg / μl), C-ABC (0.25 U / μl) or solvent that had undergone spinal cord injury surgery using the BBB scale (Basso, Beattie and Bresnahan scale) Hindlimb motor function was evaluated by administering a chemical solution containing only to the subarachnoid space. The injection amount of the drug solution was about 15 μl / day. Measurements were taken every week after injury until week 8. Measurements were made in a double blind manner.

(9) Evaluation of serotonergic fibers by immunohistochemical staining In order to evaluate the improvement of hindlimb motor function immunohistologically, staining of serotonergic fibers by counting 5-HT positive fibers I investigated. At 8 weeks after spinal cord injury, the rats were fixed by perfusion with 4% paraformaldehyde, fixed again with 4% paraformaldehyde for 1 day, and then immersed in 30% sucrose for 2 days to prepare 20 μm frozen sections. The section was reacted with a primary antibody (anti-5-HT antibody) and a secondary antibody (Alexa 488 label) and confirmed with a microscope. MetaMorph Offline version 6.3 r ² (Molecular Devices) was used for fiber count.

2. Results (1) ADAMTS-4 mRNA expression in the injured spinal cord was not significantly different before injury, 3 days after injury, 1 week, and 2 weeks in both RT-PCR and real-time RT-PCR measurements ( 1 and 2).
(2) As a result of examining the protein expression by Western blot, it was slightly elevated in the damaged group compared with the sham group (only 10th thoracic laminectomy) one week after the injury (FIG. 3). The protein activity of ADAMTS-4 by the fluorescent peptide assay was slightly enhanced by 1.4 times in the damaged group compared to the sham group (FIG. 4).
(3) When ADAMTS-4 expression was observed using mRNA collected from neurons, astrocytes and microglia after culture, stronger expression was observed in astrocytes and microglia than in neurons (FIG. 5).
(4) From the results of degradation assay using PG collected from rat brain, only ADAMTS-4 degrades blebican, neurocan and phosphacan, and inactivated ADAMTS-4 and ADAMTS-13 show no degradation activity. Was found (FIGS. 6-8). That is, it was suggested that ADAMTS-4 is specifically involved in the degradation of these proteoglycans.
(5) In the axon extension assay using ADAMTS-4, significant axon extension was observed in the ADAMTS-4 added group and the C-ABC added group compared with the control (PG alone) and ADAMTS-13 added groups. (FIG. 9). The number of cell adhesion is also significantly higher in the ADAMTS-4 added group and the C-ABC added group (FIG. 10). This result suggests that ADAMTS-4 is effective for the regeneration of the central nervous system due to its nerve axon elongation effect.
(6) In the hindlimb motor function evaluation using the BBB scale, the rats that received therapeutic intervention using ADAMTS-4 and C-ABC showed a significant improvement compared to the Vehicle group (FIG. 11). The degree of improvement was comparable between ADAMTS-4 and C-ABC with no significant difference. Thus, the therapeutic effect of ADAMTS-4 was confirmed at the animal level. The fact that ADAMTS-4 showed the same therapeutic effect as C-ABC means that if ADAMTS-4 is used, a high therapeutic effect can be obtained regardless of bacterial C-ABC. Possible treatment strategies.
(7) Compared with the vehicle group, the rats subjected to treatment intervention with ADAMTS-4 showed significantly more 5-HT positive fibers in the caudal anterior horn of the injured spinal cord (FIG. 12). That is, functional improvement was confirmed also in immunohistochemistry.

As applicable disease therapeutic agent of the present invention, spinal cord injury, myelitis, brain injury, degeneration and loss of neurons by cerebral infarction caused by cerebral ischemia or cerebral hemorrhage, and retinal diseases and the like accompanied by disorders of the nervous cells is assumed.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Sequence numbers 5 to 12: Artificial sequence description: PCR primers

Claims

A therapeutic agent for central nervous system disease comprising the following (1) or (2) as an active ingredient:
(1) ADAMTS-4 protein;
(2) An expression vector carrying the ADAMTS-4 gene.
The therapeutic agent for central nervous disease according to claim 1, wherein the ADAMTS-4 protein comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence equivalent to the amino acid sequence.
The therapeutic agent for central nervous disease according to claim 1, wherein the ADAMTS-4 gene comprises the base sequence shown in SEQ ID NO: 2 or a base sequence equivalent to the base sequence.
A disease or condition selected from the group consisting of spinal cord injury, myelitis, brain injury, neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, and retinal disease associated with neuronal damage The therapeutic agent for central nervous disease according to any one of claims 1 to 3, wherein
The therapeutic agent for central nervous disease according to any one of claims 1 to 3, wherein the central nervous disease is spinal cord injury.
A method for treating central nervous disease, comprising the step of administering a therapeutic agent for central nervous disease comprising the following (1) or (2) as an active ingredient to a patient with central nervous disease:
(1) ADAMTS-4 protein;
(2) An expression vector carrying the ADAMTS-4 gene.
The central nervous system disease is a disease selected from the group consisting of spinal cord injury, myelitis, brain injury, neuronal degeneration / dropout due to cerebral infarction associated with cerebral ischemia or intracerebral hemorrhage, and retinal disease with neuronal damage The treatment method according to claim 6.
The treatment according to claim 6, wherein the central nervous disease is spinal cord injury.